Stable expression of mucin glycoproteins GP40 and GP15 of Cryptosporidium parvum in Toxoplasma gondii

Background Cryptosporidium spp. are common protozoa causing diarrhea in humans and animals. There are currently only one FDA-approved drug and no vaccines for cryptosporidiosis, largely due to the limited knowledge of the molecular mechanisms involved in the invasion of the pathogens. Previous studies have shown that GP60, which is cleaved into GP40 and GP15 after expression, is an immunodominant mucin protein involved in the invasion of Cryptosporidium. The protein is highly O-glycosylated, and recombinant proteins expressed in prokaryotic systems are non-functional. Therefore, few studies have investigated the function of GP40 and GP15. Methods To obtain recombinant GP40 with correct post-translational modifications, we used CRISPR/Cas9 technology to insert GP40 and GP15 into the UPRT locus of Toxoplasma gondii, allowing heterologous expression of Cryptosporidium proteins. In addition, the Twin-Strep tag was inserted after GP40 for efficient purification of GP40. Results Western blotting and immunofluorescent microscopic analyses both indicated that GP40 and GP15 were stably expressed in T. gondii mutants. GP40 localized not only in the cytoplasm of tachyzoites but also in the parasitophorous vacuoles, while GP15 without the GPI anchor was expressed only in the cytoplasm. In addition, a large amount of recTgGP40 was purified using Strep-TactinXT supported by a visible band of ~ 50 kDa in SDS-PAGE. Conclusions The establishment of a robust and efficient heterologous expression system of GP40 in T. gondii represents a novel approach and concept for investigating Cryptosporidium mucins, overcoming the limitations of previous studies that relied on unstable transient transfection, which involved complex steps and high costs. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-024-06159-y.


Background
Cryptosporidium spp.primarily infect the intestinal epithelial cells of humans and a variety of animals, causing diarrhea and associated disease [1].They are transmitted through the fecal-oral route [2], and they cause cryptosporidiosis, which is the second leading cause of childhood diarrhea in middle and low-income countries and a major cause of mortality in individuals with HIV/ AIDS [3].Nitazoxanide is the only FDA-approved drug against human cryptosporidiosis, and there is no available vaccine against this infection [4].
An established strategy for the identification of potential drug targets or vaccine candidates in Cryptosporidium involves the exploration of surface antigens that participate in the recognition, attachment, and invasion of host epithelial cells, as well as the investigation of their interactions with the host [5].A family of mucin glycoproteins is involved in the invasion of Cryptosporidium into host cells [6].These mucins undergo significant O-linked glycosylation modifications, which have been shown to be crucial for their proper folding and function [7].Among them, GP60 emerges as one of the most prominent mucin proteins in the invasion of Cryptosporidium [8].This protein is cleaved into GP40 and GP15 after its expression, and GP40 remains on the parasite surface by binding to GP15, which has a GPI anchor [9,10].GP40 has > 30 O-linked glycosylation sites, while GP15 has a few such sites.In 2000, several research groups reported the discovery of the gene encoding GP60.However, few studies have investigated its function.This is mainly due to the challenges associated with conducting in-depth studies of mucins using recombinant proteins expressed in Escherichia coli, which have no biological function [9,[11][12][13].
Heterologous expression of Cryptosporidium mucins in Toxoplasma gondii probably provides recombinant proteins with the correct post-translational modifications.Toxoplasma gondii and Cryptosporidium belong to the phylum Apicomplexa, and T. gondii is amenable to in vitro cultivation [14].In addition, well-established genetic manipulation techniques such as CRISPR/Cas9 have been applied to T. gondii [15].In 2003, the transient expression of recombinant GP40/15 was achieved using the RHΔhxgprt strain of T. gondii, and mass spectrometry analysis confirmed that the recombinant GP40/15 had the same O-linked glycosylation as the native GP40/15 [16].The researchers further improved the stability of the system by modifying the expression plasmid, enabling the successful affinity purification of recombinant GP40 [17].These suggest that the heterologous expression system of T. gondii can be utilized for the expression and production of Cryptosporidium mucins.
Despite the incorporation of the episomal shuttle sequence to mitigate plasmid loss associated with transient expression of foreign proteins in T. gondii, the plasmid remains susceptible to loss during the passage, freezing, and recovery of parasites [18].In addition, the high cost of HA-tag affinity purification poses a challenge to the production of recombinant proteins for biological studies.In view of these limitations, we employed CRISPR/Cas9 technology to insert GP40 and GP15 into the UPRT locus of T. gondii, respectively [15].In addition, the Twin-Strep tag was used for protein purification, which offers improved purification efficiency at a more affordable cost [19][20][21].As a result, a more robust and efficient heterologous expression system of T. gondii was established, providing a novel approach and concept for the study of Cryptosporidium mucins.

Parasite strains
The IIdA20G1-HLJ isolate of Cryptosporidium parvum was originated from a dairy calf in Heilongjiang (HLJ) Province, China, in 2018 [22].The oocysts of IIdA20G1-HLJ were initially recovered from a fecal sample from the calf by gradient centrifugation, maintained by passage in IFN-γ knockout (GKO) mice, and stored at 4 °C in PBS containing antibiotics for < 3 months prior to use in the study [23,24].The RHΔku80 strain of T. gondii was obtained from Dr. Bang Shen at Huazhong Agricultural University in 2021.It was continuously passaged in human foreskin fibroblast (HFF, ATCC SCRC-1041, Manassas, VA, USA) monolayers and frozen in liquid nitrogen for long-time storge.Prior to use in the study, the RHΔku80 strain was recovered from the liquid nitrogen and passaged in HFF monolayers.After 2-3 days of culture, the fresh tachyzoites of the RHΔku80 strain in the HFF culture were harvested by differential centrifugation.

HFF cell culture and infection of T. gondii
The HFF cells were used for in vitro cultivation of T. gondii.The cells were seeded in flasks (T25 and T175) or plates (24-well and 96-well) and cultured in DMEM highglucose medium (Thermo, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo), 1% l-glutamine (Sigma Aldrich, St. Louis, MO, USA), and 1% antibiotic antimycotic solution (Sigma Aldrich) at 37 °C in a 5% CO 2 atmosphere until they reached approximately 80% confluence.Tachyzoites of the RHΔku80 strain or the mutants generated in the study were allowed to infect cells in confluent cultures in DMEM high-glucose medium supplemented with 2% FBS [25].

Generation of the RHΔku80 Tguprt-Cpgp40 and Tguprt-Cpgp15Δgpi mutants
Fresh tachyzoites (2 × 10 7 per transfection) of the harvested RHΔku80 strain were resuspended in 250 μl transfection buffer cytomix; 7.5 μg CRISPR/Cas9 plasmids and 1.5 μg linearized homology repair templates were added to the mixture containing tachyzoites and cytomix, which was then transferred to a cuvette (BioRad, Hercules, CA, USA) and electroporated twice (1600 V, 25 μF, 50 Ω) using a BTX Gemini system (BTX, Holliston, MA, USA).The transfected tachyzoites were allowed to infect HFF cells.After about 18 h of culture, 1 μM pyrimethamine (Life Technologies, Carlsbad, CA, USA) and 10 μM floxuridine (Thermo) were added to the culture medium for selection of the mutants.After 2-3 passages, 0-1 of tachyzoite obtained by limiting dilution was allowed to infect cells cultured in 96-well plates to obtained single colony.PCRs targeting three different sites of each mutant were used to confirm the correct integration of templates as previously described [26].Briefly, DNA was extracted from the culture of each mutant using the TIANamp Blood DNA Midi Kit (Tiagen Biotech, Beijing, China), and used as the template in the PCR analyses.The three sites of each mutant amplified by PCR are shown in Fig. 1b, and the primers used are listed in Additional file 1: Table S1.

Immunofluorescence verification of recTgGP40 and recTgGP15
The expression of C. parvum GP40 and GP15Δgpi in T. gondii was initially verified by immunofluorescence assay (IFA).For the IFA analysis of the RHΔku80 Tguprt-Cpgp40 mutant and the Tguprt-Cpgp15Δgpi mutant, HFF cells were infected with 10 5 tachyzoites of these two mutants, respectively.After 16 h of culture at 37 °C with 5% CO 2 , the HFF cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 at 37 °C for 10 min, and blocked with 1% BSA at 37 °C for 20 min.The following primary antibodies were diluted in 0.1% BSA for staining: mouse anti-HA (MBL Beijing Biotech, Beijing, China) at a 1:500 dilution, mouse anti-GP60 (purified rabbit pAbs) at 1:800 dilution.Cells were incubated with the antibodies for 40 min at 37 °C, followed by six washes with PBS.Alexa Fluor-conjugated secondary antibodies (Thermo) diluted 1:1000 and Hochest diluted 1:2000 in 0.1% BSA were added to the coverslips and incubated at 37 °C for 20 min.The cells were then washed six times with PBS and mounted on slides using Vectashield.The stained slides were examined using a Zeiss Axioskop Mot 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).Images were manipulated using ZEN microscopy software (Carl Zeiss, Oberkochen, Germany).

Western blot analysis of recTgGP40 and recTgGP15
The expression of C. parvum GP40 in T. gondii was further verified by Western blot analysis using rabbit anti-GP60 (purified rabbit pAbs).The RHΔku80 Tguprt-Cpgp40 mutant (10 5 tachyzoites) was grown in HFF cells cultured in DMEM medium for 24 h.All parasites were then purified and collected by differential centrifugation, and total proteins of the parasites were extracted using RIPA lysis buffer (Thermo) by incubation at 4 °C.The lysis was centrifugated, and 50 μl of the supernatant was used for Western blot analyses.The PVDF membrane was blocked in TBST (0.05% Tween 20 in TBS) containing 1% BSA and then incubated with the primary antibodies.The following primary antibodies were used and diluted in TBST containing 0.1% BSA: mouse anti-HA (MBL Beijing Biotech, Beijing, China) at 1:1000 dilution; rabbit anti-GP60 (purified rabbit pAbs) at 1:800 dilution.After thoroughly washing with TBST, the membrane was then incubated with HRP-conjugated antibodies (Beyotime, Shanghai, China) diluted 1:3000 in TBST containing 0.1% BSA.After washing with TBST, the membrane was incubated with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Massachusetts, USA) and analyzed using a Chemstudio imaging system (Analytik Jena, Jena, Germany).

Purification of recTgGP40
Strep-TactinXT resin (IBA GmbH, Göttingen, Germany) was used to purify recTgGP40-Twinstrep-HA as shown in Fig. 4a.In this procedure, the RHΔku80 Tguprt-Cpgp40 mutant was grown in HFF cells cultured in DMEM medium for 24 h.The cultures were then lysed with RIPA lysis buffer and centrifuged at 16,000g and 4 °C for 10 min to obtain the supernatant.The Strep-Tactin column with resin was equilibrated with wash buffer (100 mM Tris/HCl pH 8.0, 150 mM NaCl, 1 mM EDTA) and then combined with the supernatant.After the supernatant had completely entered the column, the column was successively washed with wash buffer and elution buffer (wash buffer containing 2.5 mM desthiobiotin).The eluate was further concentrated by dialysis, and 100 μl of the concentrated eluate was used for identification of protein bands by SDS-PAGE, Western blot, and liquid chromatography/mass spectrometry (LC/MS).

Construction of RHΔku80 Tguprt-Cpgp40 mutant and Tguprt-Cpgp15Δgpi mutant
For the expression of CpGP40 and CpGP15 in T. gondii, we attempted to insert the GP40 and GP15 into the UPRT locus of the RHΔku80 strain using CRISPR/Cas9 (Fig. 1b).To facilitate the purification of the recombinant protein, GP40 was truncated by removing the RSRR site, and a TwinStrep-6 × HA tag was added to the C-terminus of GP40 (Fig. 1).The HA tag was used for the identification of the mutants, and the Twin-Strep tag was used for purification of the recombinant proteins.The RHΔku80 Tguprt-Cpgp40 mutant and the RHΔku80 Tguprt-Cpgp15 mutant were obtained after selection of pyrimethamine and floxuridine, and limiting dilution.In PCR analyses of the three sites of the mutants, expected bands were present in the PCR1 and PCR2 analyses of the RHΔku80 Tguprt-Cpgp40 mutant and RHΔku80 Tguprt-Cpgp15 mutant, while no band was observed in the PCR1 and PCR2 analyses of the RHΔku80 strain (WT).This confirmed that CpGP40 and CpGP15 were correctly inserted into the UPRT locus.In the PCR3 analysis, the sequence covering the CRISPR targeting site (the UPRT locus) was amplified in the RHΔku80 strain (WT).In contrast, no bands were present in the PCR3 analysis of the RHΔku80 Tguprt-Cpgp40 mutant and the RHΔku80 Tguprt-Cpgp15 mutant, which confirmed that the UPRT was knockout in the two mutants (Fig. 2a).

Expression of GP40 in fusion with the Twin-Strep tag and HA tag
GP40 was regulated by activation of the TgGRA2 promoter, resulting in its significant overexpression.In Western blot analysis of the lysates of RHΔku80 Tguprt-Cpgp40 mutant, a ~ 50 kDa protein was recognized by HA monoclonal antibody, Strep monoclonal antibody, and GP40 monoclonal antibody, respectively (Fig. 2b).Therefore, the protein recognized was considered recTgGP40-Twinstrep-6 × HA.In addition, the size of recTgGP40-Twinstrep-6 × HA is comparable to that of the reported native GP40 with post-translational modification, indicating O-linked glycosylation of the recombinant protein.In the IFA analysis, CpGP40 was co-localized with the Twin-Strep tag and HA tag, respectively, further supporting the fusion of CpGP40 with the tags in T. gondii (Fig. 3a, b).

Distribution of recTgGP40 and recTgGP15 in T. gondii
IFA analysis of the RHΔku80 mutant (Tguprt-Cpgp40 and Tguprt-Cpgp15Δgpi) showed that recTgGP40-Twinstrep-6 × HA was distributed in both the cytoplasm of T. gondii and parasitophorous vacuole (Fig. 3a, c), while recTgGP15-6 × HA without GPI was only distributed in the cytoplasm (Fig. 3a).Because recTgGP40 has a signal peptide, it can be secreted outside the tachyzoites and into the parasitophorous vacuole.However, recTgGP15 has neither signal peptide nor GPI anchor, so it can only be distributed in the cytoplasm.This suggested two potential approaches for subsequent purification of recTgGP40.Freshly egressed tachyzoites can be lysed and used for purification of recombinant proteins.Alternatively, cells can be lysed directly prior to parasite egress, and the lysate supernatant can be collected for purification of the recombinant proteins.

Purification of recTgGP40
IFA and Western blot analyses both indicated that recTgGP40-Twinstrep-6 × HA was abundantly expressed in the cytoplasm of the RHΔku80 Tguprt-Cpgp40 mutant (Figs.3a, 4b).Therefore, freshly egressed tachyzoites of the mutant were collected and subjected to lysis for protein purification.RecTgGP40-Twinstrep-6 × HA was purified using Strep-TactinXT through the Twin-Strep tag (Fig. 4a) and then concentrated by dialysis.In SDS-PAGE and Western blot analyses of the concentrated solution, several bands were present including one of ~ 50 kDa (Fig. 4b).In the corresponding Western blot analysis, a strong band of ~ 50 kDa was recognized by HA monoclonal antibody (Fig. 4c).Because the size of the band was comparable to the predicted size of recTgGP40, we supposed the recombinant protein is recTgGP40.In the mass spectrometry analysis of the band, seven peptide sequences yielded were compatible to the GP60 sequence of IIdA20G1-HLJ isolate (Protein ID: AZJ53484.1),which confirmed the CpGP40 identity of the ~ 50 kDa band (Table 1).However, probably because of its low expression, recTgGP15 was not detectable in Western blot, and therefore could not be purified.

Discussion
GP60 is one of the most prominent mucin proteins in Cryptosporidium and is involved in the invasion of the parasites.Upon expression, it is cleaved into GP40 and GP15, with the former containing several O-linked glycosylation sites.In this study, we employed CRISPR/ Cas9 technology to insert GP40 and GP15 into the UPRT locus of T. gondii.The T. gondii mutants generated can stably express GP40 and GP15.The recombinant GP15 without the GPI anchor localized in the cytoplasm of T. gondii.In contrast, the recombinant GP40 localized in the tachyzoite cytoplasm and parasitophorous vacuole and probably underwent O-linked glycosylation in T. gondii like that in Cryptosporidium.Using Strep-TactinXT, we recovered a large amount of The mutants of T. gondii generated using CRISPR/ Cas9 technology are capable of stable expression of Cryptosporidium proteins.Toxoplasma gondii can be easily cultured and genetically manipulated in vitro, making it a suitable choice for overexpressing proteins of C. parvum, which also belongs to Apicomplexa.Previously, Cryptosporidium GP60 has been expressed heterologously in T. gondii [16].Subsequently, other Cryptosporidium mucins, P23 and Muc4, were expressed in T. gondii [27,28].However, in these studies, the heterogenous proteins were expressed through the plasmids transiently transfected into T. gondii, and the plasmids could be lost during the passages [29,30].In the current study, the heterologous genes of Cryptosporidium were integrated into the chromosome of T. gondii using CRISPR/Cas9 technology.GP40 was overexpressed in the RHΔku80 Tguprt-Cpgp40 mutants as demonstrated by Western blot and IFA analyses.Therefore, this approach has an improved stability and reliability for the overexpression of target genes of Cryptosporidium.
The recTgGP40 is probably glycosylated in a manner analogous to native expression of the protein.Native CpGP40 contains a signal peptide and several O-linked glycosylation sites [9,12].Signal peptides play crucial roles in directing proteins to the endoplasmic reticulum and Golgi apparatus for post-translational processing, including glycosylation [31,32].Therefore, the signal peptide of CpGP40 was retained during the overexpression in T. gondii in this study.The similarity in the size of recTgGP40 and native CpGP40 suggested the glycosylation of the recombinant protein in T. gondii.In addition, recTgGP40 was distributed in the cytoplasm of tachyzoites and the parasitophorous vacuoles, indicating secretion of the protein under the direction of the signal peptide.This is inconsistent with the surface localization of GP40 in C. parvum and transiently transfected T. gondii [16,33].The disruption of surface localization of recTgGP40 is probably due to the absence of GP15, which is associated with GP40 and attached to the membrane.
GP15 without a GPI anchor was only expressed in the cytoplasm.GPI is a common structural motif in apicomplexans, serving as anchors of some proteins for the attachment to cell membrane [34,35].In a previous study, GP15 was expressed in T. gondii with and without the GPI anchor [16].The result showed that GP15 was unable to localize to the membrane of tachyzoites in absence of the GPI anchor.In IFA analysis, we observed recTgGP15 in the cytoplasm rather than on the surface of tachyzoites.Therefore, the localization of GP15 on the cell membrane is indeed GPI-dependent, at least within the heterologous expression system.
Combined with the Twin-Strep-based purification, the heterologous expression system established here allowed a high yield of purified recTgGP40 at a low cost.Because C. parvum is difficult to propagate in vitro and in vivo, native GP40 can only be isolated in limited amounts from purified C. parvum oocysts.The amount of recombinant GP40 expressed by transiently transfected T. gondii was limited, which was only detectable by silver staining [17].Similarly, the heterologous expression of other C. parvum mucin proteins in T. gondii, such as P23 and Muc4, resulted in poor yields of recombinant proteins [27,28].These could be due to the loss of the plasmid containing the target genes and complicated purification procedure using a HA tag.In this study, stable heterologous expression of GP40 was achieved by inserting GP40 into the chromosome of T. gondii using CRISPR/Cas9 technology.In addition, a Twin-Strep tag was fused to the C-terminal of GP40 for purification.Compared to the purification using a HA tag, the purification using the Twin-Strep tag was achieved by specific binding between the Streptag and the Strep-Tactin affinity chromatography resin, which was more efficient and cost effective [28,36].The high efficiency of the heterologous expression and Strep-based purification was supported by a visible band of ~ 50 kDa in SDS-PAGE (Fig. 4b), which was confirmed to be recTgGP40 by LC/MS analysis.

Conclusions
In conclusion, we have established a heterologous expression system of Cryptosporidium mucin glycoproteins in T. gondii using CRISPR/Cas9 technology.The system allowed stable expression of GP40 and GP15 of C. parvum in T. gondii.In addition, we fused GP40 with a Twin-Strep-tag and HA tag, and a significant amount of recTgGP40 was purified using Strep-TactinXT resin at an affordable cost.Therefore, the novel approach and concept present here should be useful for the further study of Cryptosporidium mucins.

Fig. 1
Fig. 1 Strategy used in the heterologous expression of GP40 and GP15 of Cryptosporidium parvum in Toxoplasma gondii.a The C. parvum GP60 is composed of GP40 and GP15, with a signal peptide at the N-terminus, a GPI anchor at the C-terminus, and a furin cleavage site RSRR between the two cleavage products.In this study, GP40 was truncated and fused to the Twinstrep-6 × HA tag for expression, retaining the signal peptide of GP40 while removing the furin cleavage site RSRR.GP15 without GPI was fused to the 6 × HA tag for expression.b Schematic illustration of the CRISPR/Cas9 strategy used to generate RHΔku80 mutant (Tguprt-Cpgp40 and Tguprt-Cpgp15Δgpi) by inserting CpGP40-Twinstrep-6 × HA-DHFR* (pyrimethamine-resistant DHFR) or CpGP15Δgpi-6 × HA-DHFR*.The transfection of the sgUPRT together with an amplicon containing a CpGP40-Twinstrep-6 × HA-DHFR* (CpGP15Δgpi-6 × HA-DHFR*)-expressing cassette flanked by homology regions to UPRT was used to generate the gene insertion.The orange bar in UPRT gene represents the region targeted by the sgRNA

Fig. 2 Fig. 3 Fig. 3 (Fig. 4
Fig. 2 Verification of the correct integration and expression of the GP40 and GP15ΔGPI of Cryptosporidium parvum in Toxoplasma gondii.a Diagnostic PCR confirming the homologous integration and gene disruption in a representative clone compared to the parental line RHΔku80.PCR1 and PCR2 provide evidence of homologous integration based on products amplified between the CpGP40-Twinstrep-6 × HA-DHFR* (or CpGP15-6 × HA-DHFR*) gene and regions in the UPRT locus that lie outside of the targeting amplicon.PCR3 amplifies a 1.0-kb fragment in wild-type (WT) parasites that is lost because of the insertion of CpGP40-Twinstrep-6 × HA-DHFR* (or CpGP15Δgpi-6 × HA-DHFR*).b Expression of CpGP40-Twinstrep-6 × HA in Tguprt-Cpgp40 mutant determined by Western blotting.CpGP40 was detected with mouse anti-HA, rabbit anti-Strep, and rabbit anti-GP60, with the WT control

Table 1
Peptides of recTgGP40 matching CpGP40 obtained by LC/MS